Casein Hydrolysis Test- Objectives, Principle, Media, Procedure, Results

Objectives of Casein Hydrolysis Test

  • To determine the ability of the organism to degrade the casein protein.
  • To differentiate the organism on the basis of production of exoenzyme proteinase (caseinase)

Principle of Casein Hydrolysis Test

Casein is a macromolecule composed of amino acids linked together by peptide bonds, CO-NH. Some microorganisms have the ability to degrade the casein protein by producing proteolytic exoenzyme, called proteinase (caseinase) which breaks the peptide bond CO-NH by introducing water into the molecule, liberating smaller chains of amino acids called peptides, which are later broken down into free amino acids by extracellular or intracellular peptidases. These amino acids are transported through the cell membrane into the intracellular amino acid pool for use in the synthesis of structural and functional cellular proteins. Casein Agar or skim milk agar is a growth medium used for the detection of hydrolytic microorganisms. The agar is supplemented with skim milk as the casein source. The medium is opaque due to the casein in colloidal suspension. The hydrolysis reaction causes the milk agar, normally the opacity of real milk, to clear around the growth area as the casein protein is converted into soluble and transparent end products—small chains of amino acids, dipeptides, and polypeptides.

Media of Casein Hydrolysis Test

Skim milk agar: SM powder 28.0gm/L, Tryptone 5.0gm/L, Yeast extract 2.50gm/L, Dextrose (Glucose) 1.0gm/L, Agar 15.0gm/L, Final pH ( at 25°C)

Procedure of Casein Hydrolysis Test

  1. Inoculate the organism on the plate either a straight line or a zig-zag.
  2. Incubate the plate at 25°C or 37°C.
  3. Following incubation examine the plate for zone of hydrolysis.

Result of Casein Hydrolysis Test

Result of Casein Hydrolysis Test

Positive Test – Clearing is observed around and/or beneath colony growth (hydrolysis).

Negative Test – No clearing is observed around and/or beneath the inoculum.

Quality Control of Casein Hydrolysis Test

Bacillus subtilis ATCC 6633 – positive reaction, clear zone surrounding colonies

Pseudomonas aeruginosa ATCC 27853 – positive reaction, clear zone surrounding colonies

Escherichia coli ATCC 25922 – negative reaction, no clear zone surrounding colonies


  1. Aneja K.R. 2003. Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

1 thought on “Casein Hydrolysis Test- Objectives, Principle, Media, Procedure, Results”

  1. Good information regarding casein hydrolysis test. It would have been more better if the hydrolysis of casein is represented by chemical reaction in principle of the test.


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